Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments

AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.     

Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Occurrence and levels of indicators and selected pathogens in different sludges and biosolids

AIMS: Determine the occurrence and levels of pathogens and indicators in raw and treated sludges and compare their persistence after two different treatments. METHODS AND RESULTS: Helminth ova, Cryptosporidium spp., Salmonella spp., enteroviruses, and bacterial and viral indicators were determined in raw sludges and biosolids produced after mesophilic and thermophilic treatments. Except Salmonella, all of the parameters were quantified. Helminth ova were found at very low concentrations even in raw sludges. Viable Cryptosporidium oocysts were still present in most samples of treated sludges. Faecal coliforms, spores of sulphite-reducing clostridia (SSRC), and somatic coliphages were the only indicators with values above their detection limits in most of the samples. CONCLUSIONS: Pathogens were still detected in some treated sludge samples. SSRC were the most resistant micro-organisms to treatments and hence may be an indicator for the reduction of protozoan oocysts. Somatic coliphages constitute an alternative as viral indicators due to their detection in sludges before and after treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the persistence of some pathogens after sludge treatments, additional indicators are needed. SSRC and somatic coliphages are good candidates. Easy and inexpensive methods for the determination of these indicators are feasible both in industrialized and developing countries.     

Concentration by ultrafiltration and stabilization of phytase produced by solid-state fermentation

Ultrafiltration is an attractive downstream processing technique for concentrating enzymes and could be considered the primary step of purification. However, the efficiency of this process is often limited by protein fouling and shear-induced enzyme inactivation, which decreases permeate flux and results in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the enzyme activity was 14% during operation, indicating that the shear forces generated in the filter have significant influences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol retained 70% of the initial enzyme activity at 70 °C after 1 h and had more than 3 and 6 months storage half-life at 29 °C and 4 °C, respectively.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

Purification and biochemical characterization of a human autocrine growth factor produced by Epstein-Barr virus-transformed B cells

Autocrine growth factor for Epstein-Barr virus-transformed human B cells (aBGF), a protein that is constitutively produced by the human EBV-transformed B cell line 5/2, has been purified from serum-free conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The purified protein has a m.w. of 16,000 in NaDodSO4/polyacrylamide gel electrophoresis and an isoelectric point between 7.0 and 8.0. The relative molecular mass 16,000 form exists in equilibrium with dimeric and tetrameric forms. aBGF supports the growth of EBV-transformed B cells, which have been deprived of their own conditioned medium. The purified aBGF is fully effective at 0.5 ng/ml and has no interleukin 1 activity in the lymphocyte activation factor assay. Because several randomly selected lines of EBV-transformed cells and one EBV-negative lymphoma cell line both produce aBGF activity and show growth dependency on aBGF and because stimulation of normal B cells with anti-immunoglobulin M is increased by aBGF, we propose that aBGF has general significance for growth control of human B cells.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Characterization of a B cell-derived growth-enhancing factor produced by a human B cell line established from a patient with rheumatoid arthritis

A human B cell line, TKS-1, which was established from the peripheral blood of a patient with rheumatoid arthritis, was found to spontaneously produce a factor which enhances the activity of interleukin 1 (IL-1). This factor, designated B cell-derived growth-enhancing factor (BGEF), enhanced IL-1-induced proliferation of peanut agglutinin nonagglutinated thymocytes. BGEF also enhanced IL-1-induced production of interleukin 2 (IL-2) by both thymocytes and a human T cell clone, HSB.2 C5B2. BGEF alone did not induce the production of IL-2. BGEF failed to induce proliferation of the IL-2-dependent T cell clone, and did not enhance its response to IL-2. The activity of BGEF was not blocked by antisera against human IL-1-alpha or human IL-1-beta. Gel filtration analysis revealed that BGEF has a m.w. of 60,000 to 65,000 in its native state. We concluded that BGEF differed from IL-1 and IL-2, but is a novel factor produced by TKS-1 cells. In addition, we found that partially purified B cells from patients with rheumatoid arthritis produced factors which enhanced the activity of IL-1.     

Identification and characterization of a B cell activation factor (BCAF) produced by a human T cell line

A human T cell line, Peer, that expresses the T cell helper phenotype produces discrete activation and growth factors for tonsillar B cells. The B cell activation factor produced by Peer is biochemically and physiologically distinct from other lymphokines known to enhance B cell proliferation, namely, interleukin 1, interleukin 2, interferon, and previously characterized B cell growth factors (BCGF). The BCGF produced by Peer is functionally similar to previously described BCGF but has a m.w. of approximately 30,000 daltons. The identification and characterization of a T cell-derived activation factor that can induce apparently resting (Go phase) B cells to enter S phase in the absence of an exogenous first signal has important implications in the additional dissection of the complex steps in the human B cell cycle.     

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

Concordant evolution of mitochondrial and nuclear genomes in the wheat pathogen Phaeosphaeria nodorum

We compared patterns of mitochondrial restriction fragment length polymorphism (RFLP) diversity with patterns of nuclear RFLP diversity to investigate the effects of selection, gene flow, and sexual reproduction on the population genetic structure and evolutionary history of the wheat pathogen Phaeosphaeria nodorum. A total of 315 fungal isolates from Texas, Oregon, and Switzerland were analyzed using seven nuclear RFLP probes that hybridized to discrete loci and purified mitochondrial DNA that hybridized to the entire mtDNA genome. Forty-two different mitochondrial haplotypes and 298 different nuclear haplotypes were detected. The two most frequent mtDNA haplotypes were present in every population and represented 32% of all isolates. High levels of gene flow, low levels of population subdivision, no evidence for either host specificity or cyto-nuclear disequilibrium were inferred from the analysis of both genomes. The concordance in estimates of these population genetic parameters from both genomes suggests that the two genomes experienced similar degrees of migration, genetic drift and selection.     

Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.     

Optimization and metabolic stabilization of a class of nonsteroidal glucocorticoid modulators

The optimization of a series of nonsteroidal glucocorticoid modulators is reported. Potent selective GR ligands that have improved metabolic stability were discovered typified by the subnanomolar acid 12 (GR binding IC(50)=0.6 nM).     

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

AIM: To compare the inactivation rates of male-specific bacteriophage-2 (MS2), hepatitis A HM-175/18f (HM-175) and reovirus T1 Lang (T1 L) during alkaline stabilization of wastewater residues. METHODS AND RESULTS: A bench scale alkaline stabilization model was used to evaluate the inactivation of MS2 seeded into raw sludge simultaneously with HM-175 or T1 L. Stabilization was performed in triplicate at 28 and 4 degrees C for both viral combinations. During stabilization at 28 and 4 degrees C, MS2 and T1 L concentrations were similar at each time point (t = 0.1, 2, 12 and 24 h). MS2 and HM-175 concentrations were also similar at each time point during stabilization at 28 degrees C. At 4 degrees C, MS2 and HM-175 concentrations were not similar at the first two time points (t = 0.1 and 2 h), but were similar at later time points (t = 12 and 24 h). CONCLUSIONS: The inactivation rates of T1 L at 4 degrees C and both T1 L and HM-175 at 28 degrees C were similar to the inactivation rate of MS2 at all time points. At 4 degrees C, MS2 was inactivated at a faster rate during the first two time points (t = 0.1 and 2 h) than HM-175, but was inactivated similarly at later time points (t = 12 and 24 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Phages, such as MS2, would be ideal indicators for the presence of enteric viruses in wastewater residues because of their ubiquity, nonpathogenic nature, low cost and time associated with their detection. The findings of this study suggest that MS2 could serve as an indicator for monitoring the persistence of enteric viruses, such as HM-175 and T1 L, during alkaline stabilization performed at moderate temperatures (28 degrees C), but may not serve as an indicator for HM-175 at reduced temperature (4 degrees C). The utility of MS2 as an indicator of viral persistence during biosolids treatment should be further evaluated, as the increased efficiency and frequency of pathogen monitoring associated with their use may reduce the potential public health risk associated with biosolids, facilitating a greater acceptance for their land application.     

Fate of bacterial indicators,viruses and protozoan parasites in a wastewater multi-component treatment system

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

Unique structural properties of apolipoprotein B in low-density lipoproteins produced by several human hepatoma-derived cell lines

Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.     

Characterization and purification of acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B

In the present study, acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B, exhibited profound inhibitory activity against a variety of LAB and pathogens, including Gram-negative bacteria, and its mode of action was to destabilize the cell wall, thereby resulting in bactericidal lysis. Acidocin 1B was found to be heat stable, because it lost no activity when it was heated up to 95 degrees C for 60 min. It retained approximately 67% of the initial activity after storage for 30 days at 4 degrees C, and 50% of its initial activity after 30 days at 25 degrees C and 37 degrees C. The molecular mass of acidocin 1B was estimated to be 4214.65 Da by mass spectrometry. Plasmid curing results indicated that a plasmid, designated as pLA1B, seemed to be responsible for both acidocin 1B production and host immunity, and that the pLA1B could be transformed into competent cells of L. acidophilus ATCC 43121 by electroporation. Our findings indicate that the acidocin 1B and its producer strain may have potential value as a biopreservative in food systems.